Methods and compositions for enhancing delivery of double-stranded rna or a double-stranded hybrid nucleic acid to regulate gene expression in mammalian cells

ABSTRACT

A double-stranded RNA, preferably a small, interfering (si)RNA or a siHybrid, to which cholesterol moieties are linked.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation claiming the benefit under 35 U.S.C.§ 120 of copending U.S. patent application Ser. No. 11/107,371, filedApr. 15, 2005, which claimed the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application No. 60/564,543, filed Apr. 20, 2004, eachof which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

RNA interference is the process of sequence-specific posttranscriptional gene silencing in cells initiated by double-stranded RNA(dsRNA) that is homologous in sequence to a portion of a targeted mRNA.Introduction of dsRNA into cells leads to the destruction of theendogenous RNAs that share the same sequence as the dsRNA. The dsRNAmolecules are cleaved by an RNase III family nuclease called Dicer intoshort-interfering RNAs (siRNA), which are 19-23 nucleotides (nt) inlength. The siRNAs are incorporated into a multicomponent nucleasecomplex (RISC, RNA-induced silencing complex), which identifies mRNAsubstrates through their homology to the siRNA, binds to and destroysthe targeted mRNA. In mammalian cells, dsRNAs longer than 30 base pairscan activate the dsRNA-dependent kinase PKR and 2′-5′-oligoadenylatesynthetase, normally induced by interferon. By virtue of its small size,synthetic siRNA avoids activation of the interferon response. Theactivated PKR inhibits general translation by phosphorylation of thetranslation factor eukaryotic initiation factor 2α (eIF2α), while2′-5′-oligoadenylate synthetase causes nonspecific mRNA degradation viaactivation of RNase L.

In contrast to the nonspecific effect of long dsRNA, siRNA can mediateselective gene silencing in the mammalian system Hairpin RNA with ashort loop and 19 to 27 base pairs in the stem also selectively silencesexpression of genes that are homologous to the sequence in thedouble-stranded stem. Mammalian cells can convert short hairpin RNA intosiRNA to mediate selective gene silencing.

RISC mediates cleavage of single stranded RNA having sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA takes place in the middle of the region complementary tothe antisense strand of the siRNA duplex.

Studies have shown that 21 nucleotide siRNA duplexes are most activewhen containing two nucleotide 3′-overhangs. Furthermore, completesubstitution of one or both siRNA strands with 2′-deoxy (2′-H) or2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution ofthe 3′-terminal siRNA overhang nucleotides with deoxy nucleotides (2′-H)was shown to be tolerated.

Studies have shown that replacing the 3′-overhanging segments of a21-mer siRNA duplex having 2 nucleotide 3′ overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity.

RNA interference is emerging as a promising means for reducing theexpression of specific gene products, and thus may be useful fordeveloping therapeutic drugs to treat viral infections, cancers,autoimmune diseases, and other diseases and conditions amenable totreatment by down-regulation of mRNA expression. However, there remainsan important need in the art for additional tools and methods to design,produce, formulate, deliver, and use siRNAs as therapeutic tools,including for therapies targeted to specific tissues and cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates serum effects on cellular uptake of acholesterol-conjugated siRNA in complex with a delivery enhancing agent(comprising a permeabilizing peptide, PN73), and on an unconjugatedsiRNA in complex with PN73-expressed as percentage uptake.

FIG. 2 illustrates serum effects on cellular uptake of acholesterol-conjugated siRNA in complex with PN73, and on anunconjugated siRNA in complex with PN73—expressed as mean fluorescenceintensity (MFI).

FIG. 3 illustrates the effects of increasing concentrations of serum oncellular uptake of a cholesterol-conjugated siRNA in the presence orabsence of a second delivery enhancing agent, lipofectamine—expressed aspercentage uptake.

FIG. 4 illustrates the effects of increasing concentrations of serum oncellular uptake of a cholesterol-conjugated siRNA in the presence orabsence of a second delivery enhancing agent, lipofectamine—expressed asMFI.

DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The present invention fulfills these needs and satisfies additionalobjects and advantages by providing double-stranded nucleic acidsconjugated to a cholesterol moiety to facilitate delivery of the nucleicacids into a selected target cell or tissue. In particular the presentinvention is directed towards methods and compositions to administerdouble-stranded ribonucleic acid to a mammal so as to effectuatetransfection of the double-stranded RNA into a desired tissue of themammal. In certain embodiments the double-stranded RNA has 30 or fewernucleotides, and is a short interfering RNA (siRNA).

It has been surprisingly discovered that selectively conjugating acholesterol moiety to a siRNA at selective ends of the siRNA senseand/or antisense strands increases the silencing of the targeted mRNA.For example, the following siRNA/cholesterol moiety constructs increasethe silencing effect of the targeted mRNA in comparison to siRNA havingno cholesterol conjugated to it:

-   -   1. A siRNA construct having a cholesterol moiety linked to the        5′ end of the sense strand and the 5′end of the antisense, and        no cholesterol moiety at the other ends;    -   2. A siRNA construct having a cholesterol moiety linked to the        3′ end of the antisense strand, and no cholesterol moiety linked        to the other ends of the siRNA strands;    -   3. A siRNA construct having cholesterol moiety linked to the 5′        end of the sense strand, and no cholesterol moiety linked to the        other ends of the siRNA strands;    -   4. A siRNA construct having a cholesterol moiety linked to the        3′ end of the sense strand and no cholesterol moiety linked to        the other ends of the siRNA strands;    -   5. A siRNA construct having a cholesterol moiety linked to the        3′ end of the sense strand, a cholesterol moiety linked to the        3′ end of the antisense strand and no cholesterol moiety linked        to the other ends of the siRNA strands; and    -   6. A siRNA construct having a cholesterol moiety linked to the        5′ end of the antisense strand and no cholesterol moiety linked        to the other ends of the siRNA strands.

Thus, the constructs listed above are embodiments of the presentinvention, as well as those constructs in which the ds nucleic acid is asiHybrid in which the sense strand is a DNA molecule.

The following constructs showed a progressively decreased silencing ofthe targeted mRNA in comparison to a siRNA having no cholesterolmoieties conjugated to any of its ends:

-   1. A siRNA construct having a cholesterol moiety linked to the 3′    end of the sense strand, a cholesterol moiety linked 5′ end of the    antisense strand and no cholesterol moiety linked to the other ends    of the siRNA strands;-   2. A siRNA construct having a cholesterol moiety linked to the 3′    end of the antisense strand, a cholesterol moiety linked to the 5′    end of the antisense strand and no cholesterol moiety linked to the    other ends of the siRNA strands;-   3. A siRNA construct having a cholesterol moiety linked to the 5′    end of the sense strand a cholesterol moiety linked to the 3′ end of    the antisense strand and no cholesterol moiety linked to the other    ends of the siRNA strands;-   4. A siRNA construct having a cholesterol moiety linked to 5′ end of    the sense strand, a cholesterol moiety linked to the 3′ end of the    sense strand, and no cholesterol moiety linked to the other ends of    the siRNA strands;-   5. A siRNA construct having a cholesterol moiety linked to 5′ end of    the sense strand, a cholesterol moiety linked to the 3′ end of the    antisense strand, a cholesterol moiety linked to the 5′ end of the    antisense strand and no cholesterol moiety linked to the 3′ end of    the sense strand;-   6. A siRNA construct having a cholesterol moiety linked to 5′ end of    the sense strand, a cholesterol moiety linked to the 3′ end of the    sense strand, a cholesterol moiety linked to the 3′ end of the    antisense strand and no cholesterol moiety linked to the 5′ end of    the antisense strand;-   7. A siRNA construct having a cholesterol moiety linked to 5′ end of    the sense strand, a cholesterol moiety linked to the 3′ end of the    sense strand, a cholesterol moiety linked to the 5′ end of the    antisense strand and no cholesterol moiety linked to the 3′ end of    the antisense strand;-   8. A siRNA construct having a cholesterol moiety linked to 3′ end of    the sense strand, a cholesterol moiety linked to the 3′ end of the    sense strand, a cholesterol moiety linked to the 3′ end of the    antisense strand, a cholesterol moiety linked to the 5′ end of the    antisense strand, and no cholesterol moiety linked to the 5′ end of    the sense strand;-   9. A siRNA construct having a cholesterol moiety on the 5′ end of    the sense strand, a cholesterol moiety on the 3′ end of the sense    strand, a cholesterol moiety on the 3′ end of the antisense strand    and a cholesterol moiety on the 5′ end of the antisense strand.

DEFINITIONS

As used herein, the term “inverted repeat” refers to a nucleic acidsequence comprising a sense and an antisense element positioned so thatthey are able to form a double stranded siRNA when the repeat istranscribed. The inverted repeat may optionally include a linker or aheterologous sequence such as a self-cleaving ribozyme between the twoelements of the repeat. The elements of the inverted repeat have alength sufficient to form a double stranded RNA. Typically, each elementof the inverted repeat is about 15 to about 100 nucleotides in length,preferably about 20-30 base nucleotides, preferably about 20-25nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length.

“Silencing” refers to partial or complete loss-of-function throughtargeted inhibition of gene expression in a cell and may also bereferred to as “knock down”. Depending on the circumstances and thebiological problem to be addressed, it may be preferable to partiallyreduce gene expression. Alternatively, it might be desirable to reducegene expression as much as possible. The extent of silencing may bedetermined by any method known in the art, some of which are summarizedin International Publication No. WO 99/32619. Depending on the assay,quantitation of gene expression permits detection of various amounts ofinhibition for example, greater than 10%, 33%, 50%, 90%, 95% or 99%.

The phrase “inhibiting expression of a target gene” refers to theability of a siRNA of the invention to initiate gene silencing of thetarget gene. To examine the extent of gene silencing, samples or assaysof the organism of interest or cells in culture expressing a particularconstruct are compared to control samples lacking expression of theconstruct. Control samples (lacking construct expression) are assigned arelative value of 100%. Inhibition of expression of a target gene isachieved when the test value relative to the control is about 90%,preferably 50%, more preferably 25-0%. Suitable assays include, e.g.,examination of protein or mRNA levels using techniques known to those ofskill in the art such as dot blots, northern blots, in situhybridization, ELISA, immunoprecipitation, enzyme function, as well asphenotypic assays known to those of skill in the art.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

“Large double-stranded RNA” refers to any double-stranded RNA having asize greater than about 40 base pairs (bp) for example, larger than 100bp or more particularly larger than 300 bp. The sequence of a largedsRNA may represent a segment of a mRNA or the entire mRNA. The maximumsize of the large dsRNA is not limited herein. The double-stranded RNAmay include modified bases where the modification may be to thephosphate sugar backbone or to the nucleoside. Such modifications mayinclude a nitrogen or sulfur heteroatom or any other modification knownin the art.

The double-stranded structure may be formed by self-complementary RNAstrand such as occurs for a hairpin or a micro RNA or by annealing oftwo distinct complementary RNA strands.

“Overlapping” refers to when two RNA fragments have sequences whichoverlap by a plurality of nucleotides on one strand, for example, wherethe plurality of nucleotides (nt) numbers as few as 2-5 nucleotides orby 5-10 nucleotides or more.

“One or more dsRNAs” refers to dsRNAs that differ from each other on thebasis of sequence.

“Target gene or mRNA” refers to any gene or mRNA of interest. Any of thegenes previously identified by genetics or by sequencing can beimplemented as a target. Target genes or mRNA can include developmentalgenes and regulatory genes, as well as metabolic or structural genes orgenes encoding enzymes. The target gene may be expressed in cells inwhich a phenotype is being investigated, or in an organism in a mannerthat directly or indirectly impacts a phenotypic characteristic. Thetarget gene may be endogenous or exogenous. Such cells include any cellin the body of an adult or embryonic animal or plant including gamete orany isolated cell such as occurs in an immortal cell line or primarycell culture.

In this specification and the appended claims, the singular forms of“a”, “an” and “the” include plural reference unless the context clearlydictates otherwise.

“siRNA” means a small interfering RNA that is a short-lengthdouble-stranded RNA that are not toxic in mammalian cells. The length isnot limited to 21 to 23 bp long. There is no particular limitation inthe length of siRNA as long as it does not show toxicity. “siRNAs” canbe, for example, 15 to 49 bp, preferably 15 to 35 bp, and morepreferably 21 to 30 bp long. Alternatively, the double-stranded RNAportion of a final transcription product of siRNA to be expressed canbe, for example, 15 to 49 bp, preferably 15 to 35 bp, and morepreferably 21 to 30 bp long. The double-stranded RNA portions of siRNAsin which two RNA strands pair up are not limited to the completelypaired ones, and may contain nonpairing portions due to mismatch (thecorresponding nucleotides are not complementary), bulge (lacking in thecorresponding complementary nucleotide on one strand), and the like.Nonpairing portions can be contained to the extent that they do notinterfere with siRNA formation. The “bulge” used herein preferablycomprise 1 to 2 nonpairing nucleotides, and the double-stranded RNAregion of siRNAs in which two RNA strands pair up contains preferably 1to 7, more preferably 1 to 5 bulges. In addition, the “mismatch” usedherein is contained in the double-stranded RNA region of siRNAs in whichtwo RNA strands pair up, preferably 1 to 7, more preferably 1 to 5, innumber. In a preferable mismatch, one of the nucleotides is guanine, andthe other is uracil. Such a mismatch is due to a mutation from C to T, Gto A, or mixtures thereof in DNA coding for sense RNA, but notparticularly limited to them. Furthermore, in the present invention, thedouble-stranded RNA region of siRNAs in which two RNA strands pair upmay contain both bulge and mismatched, which sum up to, preferably 1 to7, more preferably 1 to 5 in number.

The terminal structure of siRNA may be either blunt or cohesive(overhanging) as long as siRNA enables to silence the target geneexpression due to its RNAi effect. The cohesive (overhanging) endstructure is not limited only to the 3′ overhang as reported by Tuschlet al. (ibid.), and the 5′ overhanging structure may be included as longas it is capable of inducing the RNAi effect. In addition, the number ofoverhanging nucleotides is not limited to the reported 2 or 3, but canbe any numbers as long as the overhang is capable of inducing the RNAieffect. For example, the overhang may be 1 to 8, or 2 to 4 nucleotides.Herein, the total length of siRNA having cohesive end structure isexpressed as the sum of the length of the paired double-stranded portionand that of a pair comprising overhanging single-strands at both ends.For example, in the case of 19 bp double-stranded RNA portion with 4nucleotide overhangs at both ends, the total length is expressed as 23bp. Furthermore, since this overhanging sequence has low specificity toa target gene, it is not necessarily complementary (antisense) oridentical (sense) to the target gene sequence. Furthermore, as long asthe siRNA is able to maintain its gene silencing effect on the targetgene, it may comprise a low molecular weight RNA (which may be a naturalRNA molecule such as tRNA, rRNA or viral RNA, or an artificial RNAmolecule), for example, in the overhanging portion at its one end.

In addition, the terminal structure of the “siRNA” is necessarily thecut off structure at both ends as described above, and may have astem-loop structure in which ends of one side of double-stranded RNA areconnected by a linker RNA. The length of the double-stranded RNA region(stem-loop portion) can be, for example, 15 to 49 bp, preferably 15 to35 bp, and more preferably 21 to 30 bp long. Alternatively, the lengthof the double-stranded RNA region that is a final transcription productof siRNAs to be expressed is, for example, 15 to 49 bp, preferably 15 to35 bp, and more preferably 21 to 30 bp long. Furthermore, there is noparticular limitation in the length of the linker as long as it has alength so as not to hinder the pairing of the stem portion. For example,for stable pairing of the stem portion and suppression of therecombination between DNAs coding for the portion, the linker portionmay have a clover-leaf tRNA structure. Even though the linker has alength that hinders pairing of the stem portion, it is possible, forexample, to construct the linker portion to include introns so that theintrons are excised during processing of precursor RNA into mature RNA,thereby allowing pairing of the stem portion. In the case of a stem-loopsiRNA, either end (head or tail) of RNA with no loop structure may havea low molecular weight RNA. As described above, this low molecularweight RNA may be a natural RNA molecule such as tRNA, rRNA or viralRNA, or an artificial RNA molecule.

“Antisense RNA” is an RNA strand having a sequence complementary to atarget gene mRNA, and thought to induce RNAi by binding to the targetgene mRNA. “Sense RNA” has a sequence complementary to the antisenseRNA, and annealed to its complementary antisense RNA to form siRNA.These antisense and sense RNAs have been conventionally synthesized withan RNA synthesizer.

As used herein, the term “RNAi construct” is a generic term usedthroughout the specification to include small interfering RNAs (siRNAs),hairpin RNAs, and other RNA species which can be cleaved in vivo to formsiRNAs. RNAi constructs herein also include expression vectors (alsoreferred to as RNAi expression vectors) capable of giving rise totranscripts which form dsRNAs or hairpin RNAs in cells, and/ortranscripts which can produce siRNAs in vivo. Optionally, the siRNAinclude single strands or double strands of siRNA.

An siHybrid molecule is a double-stranded nucleic acid that has asimilar function to siRNA. Instead of a double-stranded RNA molecule, asiHybrid is comprised of an RNA strand and a DNA strand. Preferably, theRNA strand is the antisense strand as that is the strand that binds tothe target mRNA. The siHybrid created by the hybridization of the DNAand RNA strands have a hybridized complementary portion and preferablyat least one 3′overhanging end.

A cholesterol moiety is a cholesterol molecule, sterol or any compoundderived from cholesterol including chlolestanol, ergosterol,stimastanol, stigmasterol, methyl-lithocholic acid, cortisol,corticosterone, Δ⁵-pregnenolone, progesterone, deoxycorticosterone,17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol,dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione,aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol,tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone,9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone,9α-fluorocortisol, testosterone, dihydrotestosterone, androstenediol,androstenedione, androstenedione, 3α,5α-androstanediol, estrone,estradiol, estrogen, spermidine cholesterol carbamate, N⁴-spermidinecholesteryl carbamate, N⁴-spermidine cholesteryl carbamate di HCl salt,N⁴-spermidine-7 dehydro cholesteryl carbamate, N4-spermine cholesterylcarbamate, N,N bis(3-aminopropyl) cholesteryl carbamate, N,Nbis(6-aminohexyl) cholesteryl carbamate, N⁴-spermidinedihydrocholesteryl carbamate, N⁴-spermidine lithocholic carbamate methylester, N¹,N⁸-bis(3-aminopropyl-N⁴-spermidine cholesteryl carbamate,N(N⁴-3-aminopropylspermidine) cholesteryl carbamate,N,N-bis(4-aminobutyl) cholesteryl carbamate, N⁴-spermidine cholesterylurea, N⁴-spermine cholesteryl urea, N⁴-spermidine dihydro cholesterylurea, N⁴-spermine dihydro cholesteryl urea,N,N-bis(N′-3-aminopropyl-N″4-aminobutyl) cholesteryl carbamate,N4spermidine cholesteryl carboxamide, and N—[N¹,N⁴,N⁸-tris(3-aminopropyl)spermidine] cholesteryl carbamate, lumisterol, cholicacid, desoxycholic acid, chenodesoxycholic acid and lithocholic acid andderivatives thereof (see, e.g., U.S. Pat. No. 6,331,524).

The following exemplary cholesterol-RNA constructs are illustrative ofvarious embodiments of the invention:

-   -   1. A siRNA or siHybrid construct having a cholesterol moiety        linked to the 5′ end of the sense strand and the 5′ end of the        antisense and no cholesterol moiety at the other ends;    -   2. A siRNA or siHybrid construct having a cholesterol moiety        linked to the 3′ end of the antisense strand and no cholesterol        moiety linked to the other ends of the siRNA or siHybrid        strands;    -   3. A siRNA or siHybrid construct having cholesterol moiety        linked to the 5′ end of the sense strand and no cholesterol        moiety linked to the other ends of the siRNA or siHybrid        strands;    -   4. A siRNA or siHybrid construct having a cholesterol moiety        linked to the 3′ end of the sense strand and no cholesterol        moiety linked to the other ends of the siRNA or siHybrid        strands;    -   5. A siRNA or siHybrid construct having a cholesterol moiety        linked to the 3′ end of the sense strand, a cholesterol moiety        linked to the 3′ end of the antisense strand and no cholesterol        moiety linked to the other ends of the siRNA or siHybrid        strands; and    -   6. A siRNA or siHybrid construct having a cholesterol moiety        linked to the 5′ end of the antisense strand and no cholesterol        moiety linked to the other ends of the siRNA or siHybrid        strands.

In more detailed embodiments of the invention, a cholesterol-conjugatedsiRNA or siHybrid is formulated with, or delivered in a coordinateadministration method with, one or more secondary delivery-enhancingagent(s) that is/are further effective to enhance delivery of thecholesterol-conjugated siRNA or siHybrid into mammalian cells. Typicallythe second delivery-enhancing agent(s) is/are effective to facilitatedelivery of the cholesterol-conjugated siRNA or siHybrid across theplasma membrane and into the cytoplasm of a targeted mammalian cell. Thetargeted cell may be any cell for which delivery of acholesterol-conjugated siRNA or siHybrid into the cell for regulation ofgene expression is desired. Exemplary target cells in this contextinclude pulmonary alveolar or other airway cells, skin cells, hepaticcells, renal cells, pancreatic cells, endothelial cells, nucleated bloodcells (e.g., lymphocytes, monocytes, macrophages, or dendritic cells),muscle cells (e.g., cardiac or smooth muscle cells), mammary cells,peripheral or central nervous system (CNS) cells, cells of the stomachor intestinal tract, tumor cells, and other cells that are amenable togene regulation for therapeutic purposes according to the methods andcompositions of the invention.

In on exemplary embodiment, the cholesterol-conjugated siRNA or siHybridare targeted for delivery to mucosal epithelial cells, for example nasalmucosal epithelial cells.

Within these and related aspects of the invention, the secondarydelivery-enhancing agent(s) may be selected from one or any combinationof the following:

(a) an aggregation inhibitory agent;

(b) a charge modifying agent;

(c) a pH control agent;

(d) a degradative enzyme inhibitory agent;

(e) a mucolytic or mucus clearing agent;

(f) a ciliostatic agent;

(g) a membrane penetration-enhancing agent selected from (i) asurfactant, (ii) a bile salt, (iii) a phospholipid additive, mixedmicelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) anNO donor compound, (vii) a long-chain amphipathic molecule (viii) asmall hydrophobic penetration enhancer; (ix) sodium or a salicylic acidderivative; (x) a glycerol ester of acetoacetic acid (xi) a cyclodextrinor beta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii)a chelating agent, (xiv) an amino acid or salt thereof, (xv) anN-acetylamino acid or salt thereof, (xvi) an enzyme degradative to aselected membrane component, (xvii) an inhibitor of fatty acidsynthesis, or (xviii) an inhibitor of cholesterol synthesis; or (xix)any combination of the membrane penetration enhancing agents recited in(g)(i)-(xix);

(h) a delivery-enhancing peptide;

(i) a vasodilator agent;

(j) a selective transport-enhancing agent; and

(k) a stabilizing delivery vehicle, carrier, support or complex-formingspecies with which the cholesterol-conjugated siRNA or siHybrid iseffectively combined, associated, contained, encapsulated or boundresulting in stabilization of the siRNA or siHybrid for enhanceddelivery.

In additional aspects of the invention, the delivery-enhancing agent(s)comprise(s) any one or any combination of two or more of the foregoingdelivery-enhancing agents recited in (a)-(k), and the formulation of thecholesterol-conjugated siRNA or siHybrid with the delivery-enhancingagents provides for increased delivery of the cholesterol-conjugatedsiRNA or siHybrid into the cytoplasm of target cells for gene regulationby the cholesterol-conjugated siRNA or siHybrid.

Any one or combination of the foregoing secondary delivery-enhancingagents may be added to a pharmaceutical composition comprising acholesterol-conjugated siRNA or siHybrid as described herein, to yield acombinatorial formulation providing greater delivery enhancement incomparison to intracellular delivery of the cholesterol-conjugated siRNAor siHybrid without the secondary delivery-enhancing agent(s).

Within coordinate administration methods of the invention, thecholesterol-conjugated siRNA or siHybrid is administered to a targetcell, tissue, or individual in combination with one or more secondarydelivery-enhancing agents in a coordinate administration protocol.Within these coordinate administration methods, thecholesterol-conjugated siRNA or siHybrid is administered to the samecell, tissue, or individual as the secondary delivery-enhancingagent(s), prior to, simultaneous with, or after administration of thesecondary delivery-enhancing agent(s), which similarly may be selectedfrom any one or combination of the following:

(a) an aggregation inhibitory agent;

(b) a charge modifying agent;

(c) a pH control agent;

(d) a degradative enzyme inhibitory agent;

(e) a mucolytic or mucus clearing agent;

(f) a ciliostatic agent;

(g) a membrane penetration-enhancing agent selected from (i) asurfactant, (ii) a bile salt, (iii) a phospholipid additive, mixedmicelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) anNO donor compound, (vii) a long-chain amphipathic molecule (viii) asmall hydrophobic penetration enhancer; (ix) sodium or a salicylic acidderivative; (x) a glycerol ester of acetoacetic acid (xi) a cyclodextrinor beta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii)a chelating agent, (xiv) an amino acid or salt thereof, (xv) anN-acetylamino acid or salt thereof, (xvi) an enzyme degradative to aselected membrane component, (xvii) an inhibitor of fatty acidsynthesis, or (xviii) an inhibitor of cholesterol synthesis; or (xix)any combination of the membrane penetration enhancing agents recited in(g)(i)-(xix);

(h) a delivery-enhancing peptide;

(i) a vasodilator agent;

(j) a selective transport-enhancing agent; and

(k) a stabilizing delivery vehicle, carrier, support or complex-formingspecies with which the cholesterol-conjugated siRNA or siHybrid iseffectively combined, associated, contained, encapsulated or boundresulting in stabilization of the siRNA or siHybrid for enhancedintracellular delivery. The coordinate administration of thecholesterol-conjugated siRNA or siHybrid and secondarydelivery-enhancing agent(s) provides for increased uptake of thecholesterol-conjugated siRNA or siHybrid into the cytoplasm of targetedcells, typically enhancing gene regulation (e.g., increasing knockdownof mRNA translation to thereby reduce expression of one or more selectedprotein(s), such as TNF-α, in the target cell.

Additional detailed description pertaining to secondarydelivery-enhancing agents, for use within the instant invention isprovided, for example, in United States Provisional Patent ApplicationsNos. 60/612,121, filed Sep. 21, 2004; 60/667,835, filed Apr. 1, 2005;60/612,285, filed Sep. 21, 2004; 60/667,871, filed Apr. 1, 2005;60/613,416, filed Sep. 27, 2004; and 60/667,833, filed Apr. 1, 2005,each incorporated herein by reference.

Within exemplary embodiments of the invention, a delivery-enhancingpeptide is employed as the secondary delivery-enhancing agent. Thedelivery-enhancing peptide may be conjugated to, combinatoriallyformulated with, or coordinately administered with, thecholesterol-conjugated siRNA or siHybrid to enhance intracellular uptakeof the cholesterol-conjugated siRNA or siHybrid and improve generegulation results achieved thereby. Delivery-enhancing peptides in thiscontext may include natural or synthetic, therapeutically orprophylactically active, peptides (comprised of two or more covalentlylinked amino acids), proteins, peptide or protein fragments, peptide orprotein analogs, peptide or protein mimetics, and chemically modifiedderivatives or salts of active peptides or proteins. Thus, as usedherein, the term “delivery-enhancing peptide” will often be intended toembrace all of these active species, i.e., peptides and proteins,peptide and protein fragments, peptide and protein analogs, peptide andprotein mimetics, and chemically modified derivatives and salts ofactive peptides or proteins. Often, the delivery-enhancing peptidecomprises a mutein that is readily obtainable by partial substitution,addition, or deletion of amino acids within a naturally occurring ornative (e.g., wild-type, naturally occurring mutant, or allelic variant)peptide or protein sequence (e.g., a sequence of a naturally occurring“cell penetrating peptide” or peptide fragment of a native protein, suchas a tight junction protein). Additionally, biologically activefragments of native peptides or proteins are included. Such mutantderivatives and fragments substantially retain the desired cellpenetrating or other delivery-enhancing activity of the correspondingnative peptide or proteins. In the case of peptides or proteins havingcarbohydrate chains, biologically active variants marked by alterationsin these carbohydrate species are also included within the invention.

The delivery-enhancing peptides, proteins, analogs and mimetics for usewithin the methods and compositions of the invention are may beconjugated to, or formulated with, the cholesterol-conjugated siRNA orsiHybrid to yield a pharmaceutical composition that includes adelivery-enhancing effective amount of the delivery-enhancing peptide,protein, analog or mimetic (i.e., an amount of the peptide sufficient todetectably enhance intracellular delivery of the cholesterol-conjugatedsiRNA or siHybrid).

Exemplary delivery-enhancing peptides for use within the methods andcompositions of the invention include any one or combination of thefollowing peptides, or active fragments, muteins, conjugates, orcomplexes thereof:

(SEQ ID NO: 1) RKKRRQRRRPPQCAAVALLPAVLLALLAP; (SEQ ID NO: 2)RQIKIWFQNRRMKWKK; (SEQ ID NO: 3) GWTLNSAGYLLGKINLKALAALAKKIL; (SEQ IDNO: 4) KLALKLALKALKAALKLA; (SEQ ID NO: 7) KLWSAWPSLWSSLWKP; (SEQ ID NO:8) AAVALLPAVLLALLAPRKKRRQRRRPPQ; (SEQ ID NO: 9)LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR (SEQ ID NO: 10)RRRQRRKRGGDIMGEWGNEIFGAIAGFLG; (SEQ ID NO: 11)KETWWETWWTEWSQPGRKKRRQRRRPPQ; (SEQ ID NO: 12) GLGSLLKKAGKKLKQPKSKRKV;and (SEQ ID NO: 13) KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ

Delivery-enhancing peptides of the invention may further include variousmodifications known in the art, e.g., for modifying the charge, membranepermeability, half-life, degradative potential, reactivity (e.g., toform conjugates), immunogenicity, or other desired properties of thesubject peptide. Exemplary modified delivery-enhancing peptides in thiscontext may include, for example, peptides modified by incorporation ofone or more selected amino- or carboxy-terminal chemical modifications.For example, amino- and/or carboxy-terminal amide, BrAc, or maleimidegroups may be included, as exemplified by the modifieddelivery-enhancing peptides shown in Table 1.

TABLE 1 Peptide Sequences Effects PN0028RKKRRQRRRPPQCAAVALLPAVLLALLAP-amide + (SEQ ID NO: 1) PN0058RQIKIWFQNRRMKWKK-amide + (SEQ ID NO: 2) PN0064BrAc-GWTLNSAGYLLGKINLKALAALAKKILamide + (SEQ ID NO: 3) PN0068BrAc-KLALKLALKALKAALKLA-amide + (SEQ ID NO: 4) PN0069 GRKKRRQRRRPQ-amide− (SEQ ID NO: 5) PN0071 RRRRRRR − (SEQ ID NO: 6) PN0228NH2-KLWSAWPSLWSSLWKP-amide +/− (SEQ ID NO: 7) PN027NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide + (SEQ ID NO: 8) PN202NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-amide + (SEQ ID NO: 9) PN250NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide + (SEQ ID NO: 10) PN183NH2-KETWWETWWTEWSQPGRKKRRQRRRPPQ-amide + (SEQ ID NO: 11) PN283Maleimide-GLGSLLKKAGKKLKQPKSKRKV-amide + (SEQ ID NO: 12) PN073KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide + (SEQ ID NO: 13) Assaymedium only

The + and − notations indicated in Table 1 for the listed peptidesrelate to activity of the peptides to enhance permeation of acrossepithelial monolayers—as determined by measurement of peptide-mediatedchanges in trans-epithelial electrical resistance (TEER). A + notationindicates that the subject peptide enhances epithelial permeation ofmacromolecules. The peptides that exhibit permeation-enhancing activitycan be tested and selected according to the methods herein to determinetheir utility for enhancing delivery of cholesterol-conjugated siRNA orsiHybrid into the cytoplasm of targeted cells to enhance gene regulation

The above disclosure generally describes the present invention, which isfurther exemplified by the following examples. These examples aredescribed solely for purposes of illustration, and are not intended tolimit the scope of the invention. Although specific terms and valueshave been employed herein, such terms and values will likewise beunderstood as exemplary and non-limiting to the scope of the invention.

Example 1 Synthesis and Purification of Cholesteryl-Labeled siRNA

Synthesis of Unmodified siRNAs:

Unmodified siRNAs were synthesized according to the general strategy forsolid-phase oligonucleotide synthesis. The syntheses proceeded from the3′- to 5′-direction [current protocols in nucleic acid chemistry,chapter 3]. The first step involved attachment of a mononucleoside/tideto the surface of an insoluble solid support through a covalent bond.All unmodified siRNAs described here were synthesized starting with aCPG-bound deoxythymidine (purchased from Glen Research, Sterling Va.).The thymidine nucleoside is covalently attached to the solid supportthrough 3′-hydroxyl group using a base labile linker. Before chainelongation can proceed, the terminal-protecting group (dimethoxytrityl,DMT) on the nucleoside is removed. This exposes a free 5′-OH group wherethe next nucleotide unit can be added. An excess of reagents is used toforce the coupling reaction to occur on as many of the immobilizednucleotides as possible. After the coupling reaction, excess reagentsare washed away. The reaction is followed by a c capping step, to blockoff non-extended sites, and an oxidation step. The process ofterminal-protecting group removal and chain extension is then repeatedusing different bases until the desired sequence has been assembled.Some or all of the protecting groups may optionally be removed, and thenthe covalent attachment to the support is hydrolyzed to release theproduct. Removal of the protecting groups were carried out with 3:1mixture of concentrated ammonia:ethanol. After removal of any remainingprotecting groups, the oligonucleotide is ready for purification anduse.

RNA syntheses were carried out by Applied Biosystems 3400 using standardphosphoramidite chemistry. The corresponding building blocks,5′-dimethoxytrityl-N-benzoyladenosine-2′-O-(t-butyldimethylsilyl)-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Bz-A-CE phosphoramidite) (I),5′-dimethoxytrityl-N-dimethylformamidine-guanosine,2′-O-(t-butyldimethylsilyl)-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(dmf-G-CE phosphoramidite) (II),5′-dimethoxytrytiyl-N-acetylcytidine-2′-O-(t-butyldimethylsilyl)-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Ac-C-CE phosphoramidite) (III),5-dimethoxytrityluridine-2′-O-(t-butyldimethylsilyl)-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(U-CE phosphoramidite) (IV) and5′-dimethoxytrityl-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(V) were purchased from Glen Research Inc. (Sterling Va.). Forun-modified sequences, the syntheses started on Controlled Pore glass(CPG) bound deoxytimidine (VI) (Applied Biosystems, Foster City, Calif.)in 0.2 or 1.0 μmol scale. Other reagents and solvents were purchasedfrom Glen Research (Sterling, Va.) and/or Applied Biosystems (FosterCity, Calif.).

DMT=4,4′-dimethoxytrytyl

Commonly used protected phosphoramidites for the synthesis of RNA

Synthesis of 3′-cholesteryl-Labeled siRNA

The synthesis of 3′-cholestery-labelled siRNAs was carried out using themodified support strategy. In this method a new modified solid phasesynthesis support must be prepared for each 3′-reporter group orconjugate. The solid phase support for attaching cholesteryl group tothe 3′-termini of oligonucleotides is commercially available. Thesynthesis of the 3′-cholesteryl-labelled oligonucleotides wereaccomplished using1-dimethoyxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-succinoyl-long-chain-alkylamino-CPG(VII, Glen Research, Sterling Va.). The designed 21 nucleotide sequencewas then assembled on this modified solid support using standardphosphoramidite protocols for RNA synthesis as described herein above.

Synthesis of 5′-cholesteryl-Labeled siRNA

A protected oligonucleotide with a free hydroxyl group at the 5′-end,immobilized on the solid support, may easily be obtained by solid phasesynthesis using either methodologies described herein above. The5′-terminal hydroxyl can then be reacted with phosphoramidites.Phosphoramidites often obtained from a molecule having a hydroxylfunctionality allow the direct introduction of a functional group orligand to the chain after oxidation and deprotection. To incorporated acholesteryl group to the 5′-end of siRNA molecules,dimethoxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(VIII) was purchased from Glen Research (Sterling, Va.). During thesolid support synthesis of siRNA, after the incorporation of the lastnucleoside/tide, the 5′-dimethoyxtrytyl protecting group was cleaved andVIII was coupled to the grown chain

1-dimethoyxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-succinoyl-long-chain-alkylamino-CPG(VII)

dimethoxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(VIII)Syntheses of 3′ and 5′-dicholesteryl-Labeled siRNAs

Syntheses of 3′,5′-dicholesteryl-labeled siRNAs were accomplished usinga combination of the methods described above. The synthesis of such amolecule started with using VII as the “modified solid support”, andelongation and incorporation of the 5′-cholesteryl moiety were carriedout as described above.

Example 2 Cholesterol-Enhanced Uptake of siRNA and Silencing ofBeta-Galactosidase mRNA Expression Transfection of 9 L/LacZ Cells: Day0:

-   -   a) Take saturated 9 L/LacZ culture from T75 flask, detach cell        and dilute into 10 ml with complete medium (DMEM, 1×PS, 1×Na        Pyruvate, 1×NEAA).    -   b) Further dilute the cell to 1:15, and seed 100 μl into each 96        well, which should give 50% confluence cell the next day for        transfection. Remember to leave the edge well empty and fill        with 250 μl water, do not stack up plates in the incubator.    -   c) Incubate overnight at 37° C., 5% CO₂ incubator.

Day 1:

-   -   a) Prepare the transfection complex in Opti-MEM, 50 μl each        well.    -   b) Dump the medium in plates, wash each well once with 200 μl        PBS or Opti-MEM.    -   c) Blot the plates dry completely with tissue by inversion.    -   d) Add the transfection mixture (50 μl/well) into each well, add        250 μl water into wells on the edge to prevent wells from        drying.    -   e) Incubate for at least 3 hours at 37° C., 5% CO₂ incubator.    -   f) Dump the transfection mixture, replace with 100 μl of        complete medium (DMEM, 1×PS, 1×Na Pyruvate, 1×NEAA).

β-Gal/BCA Assay in 96 Well Format Cell Lysis

-   -   a) Dump the medium, wash once with 200 μl PBS, blot the plate        dry with inversion.    -   b) Add 30 μl lysis buffer from β-Gal Kit into each well.    -   c) Freeze-Thaw the cells twice to generate lysate.        β-Gal assay    -   a) Prepare assay mix (50 μl 1× buffer, 17 μl ONPG each well)    -   b) Take new plate and add 65 ul assay mix into each well.    -   c) Add 10 μl of cell lysate into each well. There should be        blank wells for subtraction of the background activities.    -   d) Incubate at 37° C. for about 20 minutes, prevent long        incubation which will use up all ONPG and biased the high        expression.    -   e) Add 100 μl of the Stop solution.    -   f) Measure the OD at 420 nm.

BCA Assay

-   -   a) Prepare BSA standard (150 ul per well), every points should        be duplicated on each plate.    -   b) Put 145 μl of water into each well, add 5 ul of cell lysate        into each well.    -   c) Prepare Assay Reagent (A:B:C: 25:24:1), mix right before use.    -   d) Add 150 μl of Assay Reagent into each well.    -   e) Incubate at 37° C. for about 20 minutes.    -   f) Measure the OD at 562 nm.        Flow Cytometry Measurement of FITC/FAM Conjugated siRNA    -   a) After transfection, incubate cell for at least 3 hours.    -   b) Wash with 200 μl PBS.    -   c) Detach cell with 15 μl TE, incubate at 37° C.    -   d) Re-suspend five wells with 30 μl FACS solution (PBS with 0.5%        BSA, and 0.1% sodium Azide)    -   e) Combine all five wells into a tube.    -   f) Add PI 5 μl into each tube.    -   g) Analyze the cells with fluorescence activated cell sorting        (FCAS) with BD FACscan instrument according to manufacture's        instruction.

Results

Cholesterol Conjugation of siRNA

The transfection was performed with either regular siRNA orcholesterol-conjugated siRNA with lipofectamine (Invitrogen) on 9L/beta-gal cells. The siRNA was designed to specifically knock downbeta-galactosidase mRNA and activities are expressed as percentage ofbeta-gal activities from control (transfected cells by lipofectaminealone).

1. siRNA Sequence and Structure Information of Cholesterol-ConjugatedsiRNA

C.U.A.C.A.C.A.A.A.U.C.A.G.C.G.A.U.U.U.dT.dT (Sense) (SEQ ID NO: 14)

A.A.A.U.C.G.C.U.G.A.U.U.U.G.U.G.U.A.G.dT.dT (Antisense) (SEQ ID NO: 15)

Designation of Cholesterol Conjugated siRNA

A. regular sense or antisense strand

B. 5′ end labeled sense strand

C. 3′ end labeled sense strand

D. both ends labeled sense strand

E. 5′ end labeled antisense strand

F. 3′ end labeled antisense strand

G. both end labeled antisense strand

TABLE 1 Cholesterol siRNA Activities Post-Transfection Activity (% ofActivity (% of Duplexes control Duplexes control AA 23.12 AG 29.87 BE10.27 BF 32.02 AF 11.99 DA 33.99 BA 12.09 BG 46.39 CA 16.18 DF 65.4 CF16.76 DE 77.12 AE 19.02 CG 77.80 CE 27.62 DG 98.84

Table 1 above provides results of transfection and mRNA silencingexperiments using the siRNA constructs made using sense and antisensestrands designated above. The transfection and silencing assay resultsshow cholesterol-enhanced delivery of exemplary siRNAs of the invention,and demonstrate silencing of the beta-galactosidase mRNA by thecholesterol-conjugated siRNAs. The “Activity (% of control)” indicatesthe beta-galactosidase activity remaining after the transfection. Thelower the percentage, the greater was the efficacy of the siRNAconstruct. The double letters represent a double-stranded siRNA. Thus,the exemplary constructs, BE, AF, BA, CA, CF, and AE are representativeof the nature and activity of cholesterol conjugated dsRNAs of thepresent invention. These constructs show greater silencing efficacy thanthe corresponding unconjugated siRNAs. siRNA constructs CE, AG, BF, DA,BG, DF, DE, CG and DG showed lower efficacy than the unconjugated siRNAconstruct AA.

AA is a siRNA construct with no cholesterol conjugated to any of theends of the sense or antisense RNA strands. This construct wastransfected into the cells resulting in silencing of thebeta-galactosidase mRNA so that 23.12% of the activity of thebeta-galactosidase mRNA remained.

BE is a siRNA construct having a cholesterol moiety linked to the 5′ endof the sense strand and a cholesterol moiety linked to the 5′ end of theantisense strand, and no cholesterol moiety linked to the other ends ofthe siRNA. This construct was transfected into the cells resulting insilencing the beta-galactosidase mRNA so that only 10.27% of theactivity of the beta-galactosidase mRNA remained. This is unexpectedlysuperior to the unconjugated siRNA.

AF is a siRNA construct having a cholesterol moiety linked to the 3′ endof the antisense strand and no cholesterol moiety linked to the otherends of the siRNA strands. This construct was transfected into the cellsresulting in silencing the beta-galactosidase mRNA so that only 11.99%of the activity of the beta-galactosidase mRNA remained. This isunexpectedly superior to the unconjugated siRNA.

BA is a siRNA construct having a cholesterol moiety linked to the 5′ endof the sense strand and no cholesterol moiety linked to the other endsof the siRNA strands. This construct was transfected into the cellsresulting in silencing the beta-galactosidase mRNA so that only 12.09%of the activity of the beta-galactosidase mRNA remained. This isunexpectedly superior to the unconjugated siRNA.

CA is a siRNA construct having a cholesterol moiety linked to the 3′ endof the sense strand and no cholesterol moiety linked to the other endsof the siRNA strands. This construct was transfected into the cellsresulting in silencing the beta-galactosidase mRNA so that only 16.18%of the activity of the beta-galactosidase mRNA remained. This isunexpectedly superior to the unconjugated siRNA.

CF is a siRNA construct having a cholesterol moiety linked to the 3′ endof the sense strand, a cholesterol moiety linked to the 3′ end of theantisense strand, and no cholesterol moiety linked to the other ends ofthe siRNA strands. This construct was transfected into the cellsresulting in silencing the beta-galactosidase mRNA so that only 16.76%of the activity of the beta-galactosidase mRNA remained. This isunexpectedly superior to the unconjugated siRNA.

AE is a siRNA construct having a cholesterol moiety linked to the 5′ endof the antisense strand and no cholesterol moiety linked to the otherends of the siRNA strands. This construct was transfected into the cellsresulting in silencing the beta-galactosidase mRNA so that only 19.02%of the activity of the beta-galactosidase mRNA remained. This isunexpectedly superior to the unconjugated siRNA.

The constructs listed below showed lower ability to silence thebeta-galactosidase reporter than was determined for the corresponding,unconjugated siRNA.

CE is a siRNA construct having a cholesterol moiety linked to the 3′ endof the sense strand, a cholesterol moiety linked 5′ end of the antisensestrand, and no cholesterol moiety linked to the other ends of the siRNAstrands. This construct was transfected into the cells resulting insilencing the beta-galactosidase mRNA so that 27.62% of the activity ofthe beta-galactosidase mRNA remained. This silencing effect was lowerthan that observed for the corresponding, unconjugated siRNA.

AG is a siRNA construct having a cholesterol moiety linked to the 3′ endof the antisense strand, a cholesterol moiety linked to the 5′ end ofthe antisense strand, and no cholesterol moiety linked to the other endsof the siRNA strands. This construct was transfected into the cellsresulting in silencing the beta-galactosidase mRNA so that 29.87% of theactivity of the beta-galactosidase mRNA remained. This silencing effectwas lower than that observed for the corresponding, unconjugated siRNA.

BF is a siRNA construct having a cholesterol moiety linked to the 5′ endof the sense strand, a cholesterol moiety linked to the 3′ end of theantisense strand, and no cholesterol moiety linked to the other ends ofthe siRNA strands. This construct was transfected into the cellsresulting in silencing the beta-galactosidase mRNA so that 32.02% of theactivity of the beta-galactosidase mRNA remained. This silencing effectwas lower than that observed for the corresponding, unconjugated siRNA.

DA is a siRNA construct having a cholesterol moiety linked to 5′ end ofthe sense strand, a cholesterol moiety linked to the 3′ end of the sensestrand, and no cholesterol moiety linked to the other ends of the siRNAstrands. This construct was transfected into the cells resulting insilencing the beta-galactosidase mRNA so that 33.99% of the activity ofthe beta-galactosidase mRNA remained. This silencing effect was lowerthan that observed for the corresponding, unconjugated siRNA.

BG is a siRNA construct having a cholesterol moiety linked to 5′ end ofthe sense strand, a cholesterol moiety linked to the 3′ end of theantisense strand, a cholesterol moiety linked to the 5′ end of theantisense strand, and no cholesterol moiety linked to the 3′ end of thesense strand. This construct was transfected into the cells resulting insilencing the beta-galactosidase mRNA so that 46.39% of the activity ofthe beta-galactosidase mRNA remained. This silencing effect was lowerthan that observed for the corresponding, unconjugated siRNA.

DF is a siRNA construct having a cholesterol moiety linked to 5′ end ofthe sense strand, a cholesterol moiety linked to the 3′ end of the sensestrand, a cholesterol moiety linked to the 3′ end of the antisensestrand, and no cholesterol moiety linked to the 5′ end of the antisensestrand. This construct was transfected into the cells resulting insilencing the beta-galactosidase mRNA so that 65.40% of the activity ofthe beta-galactosidase mRNA remained. This silencing effect was lowerthan that observed for the corresponding, unconjugated siRNA.

DE is a siRNA construct having a cholesterol moiety linked to 5′ end ofthe sense strand, a cholesterol moiety linked to the 3′ end of the sensestrand, a cholesterol moiety linked to the 5′ end of the antisensestrand and no cholesterol moiety linked to the 3′ end of the antisensestrand. This construct was transfected into the cells resulting insilencing the beta-galactosidase mRNA so that 77.12% of the activity ofthe beta-galactosidase mRNA remained. This silencing effect was lowerthan that observed for the corresponding, unconjugated siRNA.

BG is a siRNA construct having a cholesterol moiety linked to 3′ end ofthe sense strand, a cholesterol moiety linked to the 3′ end of the sensestrand, a cholesterol moiety linked to the 3′ end of the antisensestrand, a cholesterol moiety linked to the 5′ end of the antisensestrand, and no cholesterol moiety linked to the 5′ end of the sensestrand. This construct was transfected into the cells resulting insilencing the beta-galactosidase mRNA so that 77.80% of the activity ofthe beta-galactosidase mRNA remained. This silencing effect was lowerthan that observed for the corresponding, unconjugated siRNA.

DG is a siRNA construct having a cholesterol moiety on the 5′ end of thesense strand, a cholesterol moiety on the 3′ end of the sense strand, acholesterol moiety on the 3′ end of the antisense strand, and acholesterol moiety on the 5′ end of the antisense strand. This constructwas transfected into the cells resulting in silencing thebeta-galactosidase mRNA so that 98.84% of the activity of thebeta-galactosidase mRNA remained. This silencing effect was lower thanthat observed for the corresponding, unconjugated siRNA.

Example 3 Serum Inhibition of Cholesterol-Enhanced siRNA Uptake, andRescue of Cholesterol Enhancement of Uptake by AdditionalDelivery-Enhancing Agents Human Monocyte Isolation and Purity

Fresh human blood samples from healthy donors were purchased from GoldenWest Biologicals (Temecula, Calif.). For isolation of monocytes, bloodsamples were diluted with PBS at 1:1 ratio immediately after receiving.Peripheral blood mononuclear cells (PBMC) were first isolated by Ficoll(Amersham, Calif., USA) gradient from whole blood. Then monocytes werefurther purified from PBMCs using Miltenyi CD14 positive selection kit(MILTENYI BIOTEC GmbH, Germany) by following the manufacturer'sinstructions. The purity of the monocytes was greater than 95%, judgedby flow cytometry stained with anti-CD14 antibody (BD Biosciences, CA).Purified human monocytes were maintained overnight in complete mediabefore induction and knockdown assay.

Flow Cytometry

Fluorescence activated cell sorting (FACS) analysis were performed usingBeckman Coulter FC500 cell analyzer (Fullerton, Calif.). The instrumentwas adjusted according to the fluorescence probes used (FAM or Cy5 forsiRNA and FITC and PE for CD14). Propidium iodide (Fluka, St Lois, Mo.)and AnnexinV (R&D systems, Minneapolis, Minn.) were used as indicatorsfor cell viability and cytotoxicity.

For siRNA uptake analysis, cells were washed with PBS, treated withtrypsin (attached cells only), and then analyzed by flow cytometry.Uptake of the siRNA designated BA, described above, was also measured byintensity of Cy5 or FAM fluorescence in the cells and cellular viabilityassessed by addition of propidium iodide or AnnexinV-PE. In order todifferentiate the cellular uptake from the membrane insertion offluorescence labeled siRNA, trypan blue was used to quench thefluorescence on the cell membrane surface.

TABLE 2 Higher MFI with PN73 compared with cholesterol siRNA aloneUnconjugated siRNA with Serum Cholesterol siRNA alone 20 μM PN73  0 24.8 32.9  5% 1.55 11.5 10% 1.34 6.39 20% 1.19 5.85

The data in Table 2 show that the presence of serum significantlyreduces cellular uptake of the siRNA conjugated to a cholesterol moietyaccording to the invention. Serum also inhibits unconjugated siRNAuptake in the presence of an exemplary delivery-enhancing peptide, PN73

(KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide; SEQ ID NO: 13),but to a lesser extent than the inhibition noted for thecholesterol-conjugated siRNA.

FIGS. 1 and 2 illustrate the effects of 5% serum on cellular uptake of acholesterol-conjugated siRNA according to the invention in complex witha permeabilizing peptide delivery enhancing agent, PN73 (cholesterolsiRNA+PN73), and on an unconjugated siRNA in complex with PN73(siRNA+PN73). For these and related uptake assays,cholesterol-conjugated siRNA and siRNA/PN73 complex were transfectedinto human monocytes in Opti-MEM® media (Invitrogen) as described above,with serum added in fixed or varied concentration(s). The finalconcentration of siRNA for both cholesterol and complex were 0.2 μM. Theuptake efficiency and Mean fluorescence intensity were assessed by flowcytometry. The cellular uptake values shown in FIGS. 1 and 2 weredetermined with variation of PN73 concentrations in the presence of afixed, 5% concentration of serum.

FIGS. 3 and 4 illustrate the effects of varying concentrations of serumon cellular uptake of a cholesterol-conjugated siRNA in the presence orabsence of a second delivery enhancing agent, lipofectamine, asdetermined by flow cytometry.

The foregoing studies demonstrate that cholesterol-conjugation of siRNAscan significantly enhance their cellular uptake. However, uptake ofcholesterol-conjugated siRNAs can be substantially diminished or eveneliminated by the presence of serum. This is likely due to binding ofthe cholesterol moiety with serum proteins—inhibiting the ability of thecholesterol-bound siRNAs to enter target cells. In the presence of aselected delivery enhancing agent, Lipofectamine, this inhibitory effectof serum on cholesterol-siRNA uptake can be effectively diminished. Inaddition, the presence of a different kind of delivery enhancing agent,exemplified by the permeabilizing peptide PN73, can also mediate rescueof siRNA delivery blocked by serum. More specifically, the addition of apermeabilizing peptide to a delivery formulation comprising a siRNAconjugated to a cholesterol moiety reduces the inhibitory effects ofserum on cholesterol-siRNA uptake in a dose dependent manner. Thisdiscovery indicates that, although cholesterol conjugation to siRNAalone may not optimize siRNA delivery, additional delivery-enhancingagents including, but not limited to, Lipofectamine and PN73, canfurther enhance siRNA delivery to mammalian cells and tissues in vitroand in vivo.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications may be practicedwithin the scope of the appended claims which are presented by way ofillustration not limitation. In this context, various publications andother references have been cited within the foregoing disclosure foreconomy of description. Each of these references is incorporated hereinby reference in its entirety for all purposes. It is noted, however,that the various publications discussed herein are incorporated solelyfor their disclosure prior to the filing date of the presentapplication, and the inventors reserve the right to antedate suchdisclosure by virtue of prior invention.

1. A double-stranded RNA which is active for RNA interference having a sense and an anti-sense strand, each strand having a 5′ end and a 3′ end, wherein a cholesterol moiety is linked to the 5′ end of the antisense strand.
 2. The double-stranded RNA of claim 1 having a length of 19 to 21 base pairs.
 3. The double-stranded RNA of claim 1, wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, a compound derived from cholesterol, chlolestanol, ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid, cortisol, corticosterone, Δ⁵-pregnenolone, progesterone, deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone, 9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone, 9α-fluorocortisol, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3α,5α-androstanediol, estrone, estradiol, estrogen, spermidine cholesterol carbamate, N⁴-spermidine cholesteryl carbamate, N⁴-spermidine cholesteryl carbamate di HCl salt, N⁴-spermidine-7 dehydro cholesteryl carbamate, N⁴-spermine cholesteryl carbamate, N,N bis(3-aminopropyl) cholesteryl carbamate, N,N bis(6-aminohexyl) cholesteryl carbamate, N⁴-spermidine dihydrocholesteryl carbamate, N⁴-spermidine lithocholic carbamate methyl ester, N¹,N⁸-bis(3-aminopropyl-N⁴-spermidine cholesteryl carbamate, N(N⁴-3aminopropylspermidine) cholesteryl carbamate, N,N-bis(4-aminobutyl) cholesteryl carbamate, N⁴-spermidine cholesteryl urea, N⁴-spermine cholesteryl urea, N⁴-spermidine dihydro cholesteryl urea, N⁴-spermine dihydro cholesteryl urea, N,N-bis(N′-3-aminopropyl-N″4-aminobutyl) cholesteryl carbamate, N4spermidine cholesteryl carboxamide, and N—[N¹,N⁴,N⁸-tris (3-aminopropyl)spermidine] cholesteryl carbamate, lumisterol, cholic acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid, and derivatives thereof.
 4. An siHybrid having a sense strand and an antisense strand, each strand having a 5′ end and a 3′ end, wherein a cholesterol moiety is linked to the 3′ end of the antisense strand.
 5. The siHybrid of claim 4 having a length of 19 to 21 base pairs.
 6. The siHybrid of claim 4, wherein the sense strand is a DNA strand.
 7. The siHybrid of claim 4, wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, a compound derived from cholesterol, chlolestanol, ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid, cortisol, corticosterone, Δ⁵-pregnenolone, progesterone, deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone, 9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone, 9α-fluorocortisol, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3α,5α-androstanediol, estrone, estradiol, estrogen, spermidine cholesterol carbamate, N⁴-spermidine cholesteryl carbamate, N⁴-spermidine cholesteryl carbamate di HCl salt, N⁴-spermidine-7 dehydro cholesteryl carbamate, N⁴-spermine cholesteryl carbamate, N,N bis(3-aminopropyl) cholesteryl carbamate, N,N bis(6-aminohexyl) cholesteryl carbamate, N⁴-spermidine dihydrocholesteryl carbamate, N⁴-spermidine lithocholic carbamate methyl ester, N¹,N⁸-bis(3-aminopropyl-N⁴-spermidine cholesteryl carbamate, N(N⁴-3aminopropylspermidine) cholesteryl carbamate, N,N-bis(4-aminobutyl) cholesteryl carbamate, N⁴-spermidine cholesteryl urea, N⁴-spermine cholesteryl urea, N⁴-spermidine dihydro cholesteryl urea, N⁴-spermine dihydro cholesteryl urea, N,N-bis(N′-3-aminopropyl-N″4-aminobutyl) cholesteryl carbamate, N4spermidine cholesteryl carboxamide, and N—[N¹,N⁴,N⁸-tris (3-aminopropyl)spermidine] cholesteryl carbamate, lumisterol, cholic acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid, and derivatives thereof.
 8. A composition for delivering a double-stranded RNA to a mammalian cell comprising a double-stranded RNA having a sense and an anti-sense strand, each strand having a 5′ end and a 3′ end, wherein a cholesterol moiety is linked to the 5′ end of the antisense strand, and one or more delivery-enhancing agents selected from: (a) an aggregation inhibitory agent; (b) a charge modifying agent; (c) a pH control agent; (d) a degradative enzyme inhibitory agent; (e) a mucolytic or mucus clearing agent; (f) a ciliostatic agent; (g) a membrane penetration-enhancing agent selected from (i) a surfactant, (ii) a bile salt, (iii) a phospholipid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) an NO donor compound, (vii) a long-chain amphipathic molecule (viii) a small hydrophobic penetration enhancer; (ix) sodium or a salicylic acid derivative; (x) a glycerol ester of acetoacetic acid (xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii) a chelating agent, (xiv) an amino acid or salt thereof, (xv) an N-acetylamino acid or salt thereof, (xvi) an enzyme degradative to a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, or (xviii) an inhibitor of cholesterol synthesis; or (xix) any combination of the membrane penetration enhancing agents recited in (i)-(xviii); (h) a delivery-enhancing peptide; (i) a vasodilator agent; (j) a selective transport-enhancing agent; and (k) a RNA-stabilizing vehicle or carrier.
 9. The composition of claim 8, wherein the double-stranded RNA has a length of 19 to 21 base pairs.
 10. The composition of claim 8, wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, a compound derived from cholesterol, chlolestanol, ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid, cortisol, corticosterone, Δ⁵-pregnenolone, progesterone, deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone, 9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone, 9α-fluorocortisol, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3α,5α-androstanediol, estrone, estradiol, estrogen, spermidine cholesterol carbamate, N⁴-spermidine cholesteryl carbamate, N⁴-spermidine cholesteryl carbamate di HCl salt, N⁴-spermidine-7 dehydro cholesteryl carbamate, N4-spermine cholesteryl carbamate, N,N bis(3-aminopropyl) cholesteryl carbamate, N,N bis(6-aminohexyl) cholesteryl carbamate, N⁴-spermidine dihydrocholesteryl carbamate, N⁴-spermidine lithocholic carbamate methyl ester, N¹,N⁸-bis(3-aminopropyl-N⁴-spermidine cholesteryl carbamate, N(N⁴-3aminopropylspermidine) cholesteryl carbamate, N,N-bis(4-aminobutyl) cholesteryl carbamate, N⁴-spermidine cholesteryl urea, N⁴-spermine cholesteryl urea, N⁴-spermidine dihydro cholesteryl urea, N⁴-spermine dihydro cholesteryl urea, N,N-bis(N′-3-aminopropyl-N″4-aminobutyl) cholesteryl carbamate, N4spermidine cholesteryl carboxamide, and N—[N¹,N⁴,N⁸-tris (3-aminopropyl)spermidine] cholesteryl carbamate, lumisterol, cholic acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid, and derivatives thereof.
 11. The composition of claim 8, wherein the delivery-enhancing agent is a delivery-enhancing peptide having the amino acid sequence selected from the group of: (SEQ ID NO: 1) RKKRRQRRRPPQCAAVALLPAVLLALLAP; (SEQ ID NO: 2) RQIKIWFQNRRMKWKK; (SEQ ID NO: 3) GWTLNSAGYLLGKINLKALAALAKKIL; (SEQ ID NO: 4) KLALKLALKALKAALKLA; (SEQ ID NO: 7) KLWSAWPSLWSSLWKP; (SEQ ID NO: 8) AAVALLPAVLLALLAPRKKRRQRRRPPQ; (SEQ ID NO: 9) LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR (SEQ ID NO: 10) RRRQRRKRGGDIMGEWGNEIFGAIAGFLG; (SEQ ID NO: 11) KETWWETWWTEWSQPGRKKRRQRRRPPQ; (SEQ ID NO: 12) GLGSLLKKAGKKLKQPKSKRKV; and (SEQ ID NO: 13) KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ.


12. The composition of claim 11, wherein the delivery-enhancing peptide is conjugated to the double-stranded RNA.
 13. The composition of claim 8, wherein the mammalian cell is selected from a pulmonary alveolar cell, a skin cell, an hepatic cell, a renal cell, a pancreatic cell, an endothelial cell, a nucleated blood cell, a muscle cell, a mammary cell, a peripheral cell, a central nervous system cell, a gastrointestinal cell, and a tumor cell. 